Using FRET sensor Rango, previously we performed two primary high throughput screens (HTS) to identify small molecule inhibitors of importin beta-importin alpha binding (Assay A) and inhibitors of RanGTP-induced importin beta dissociation from importin alpha (Assay B). However, owing to the limited personnel in the lab, most of our time and resources had to be focused on our main project in the lab, the studies of the mitotic and cell cycle functions of Ran. Since the completion of the primary screens in 2011, we adapted or developed several types of secondary screens to identify specific and selective inhibitors among the primary screen hits. Through testing several different approaches, we identified two types of assays as suitable for this purpose. The first is a live cell-based assay with a stable HEK 293T cell line expressing calcium-regulated GFP-NFAT reporter for nuclear import and export. We validated this assay using Importazole (inhibitor of importin beta-RanGTP interaction; Soderholm et al., ACS Chem. Biol. 2011; 6:700-8)) as a positive control. As the second assay for the secondary screens, we developed the bead suspension assay. In this assay, importin beta is immobilized on microbeads and its interaction with fluorescently tagged importin beta binding domain (IBB) is monitored under microscope. Since the IBB-importin beta interaction is regulated by RanGTP, the disruption of RanGTP binding to importin by an inhibitor is visualized via persistent bead-bound fluorescence. We validated this assay with importazole, using 384-well glass bottom plates that would be suitable for high throughput screening.